EP3283466A1 - Antagonistes de la kinase ksr - Google Patents

Antagonistes de la kinase ksr

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Publication number
EP3283466A1
EP3283466A1 EP16780908.6A EP16780908A EP3283466A1 EP 3283466 A1 EP3283466 A1 EP 3283466A1 EP 16780908 A EP16780908 A EP 16780908A EP 3283466 A1 EP3283466 A1 EP 3283466A1
Authority
EP
European Patent Office
Prior art keywords
group
compound
pharmaceutically acceptable
acceptable salt
halo
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP16780908.6A
Other languages
German (de)
English (en)
Other versions
EP3283466A4 (fr
Inventor
Arvin Dar
Neil DHAWAN
Alex P. SCOPTON
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Icahn School of Medicine at Mount Sinai
Original Assignee
Icahn School of Medicine at Mount Sinai
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Filing date
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Application filed by Icahn School of Medicine at Mount Sinai filed Critical Icahn School of Medicine at Mount Sinai
Publication of EP3283466A1 publication Critical patent/EP3283466A1/fr
Publication of EP3283466A4 publication Critical patent/EP3283466A4/fr
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/70—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings condensed with carbocyclic rings or ring systems
    • C07D239/72—Quinazolines; Hydrogenated quinazolines
    • C07D239/74—Quinazolines; Hydrogenated quinazolines with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, attached to ring carbon atoms of the hetero ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/70—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings condensed with carbocyclic rings or ring systems
    • C07D239/72—Quinazolines; Hydrogenated quinazolines
    • C07D239/86—Quinazolines; Hydrogenated quinazolines with hetero atoms directly attached in position 4
    • C07D239/88—Oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/70—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings condensed with carbocyclic rings or ring systems
    • C07D239/72—Quinazolines; Hydrogenated quinazolines
    • C07D239/86—Quinazolines; Hydrogenated quinazolines with hetero atoms directly attached in position 4
    • C07D239/93—Sulfur atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/70—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings condensed with carbocyclic rings or ring systems
    • C07D239/72—Quinazolines; Hydrogenated quinazolines
    • C07D239/86—Quinazolines; Hydrogenated quinazolines with hetero atoms directly attached in position 4
    • C07D239/94—Nitrogen atoms

Definitions

  • This invention relates to antagonists of Kinase Suppressor of Ras (KSR) and more particularly to KSR inhibitors that are useful for the treatment of cancer.
  • Ras-mutant cancers unlike RAF -mutant cancers, it has thus far not been possible to inhibit the MAPK pathway sufficiently using therapeutically safe doses of RAF and MEK inhibitors partly due to the limited therapeutic window offered by these drug targets.
  • One in four of all human cancers contain mutant forms of Ras and Ras is the one of the most frequently mutated oncogenes (see e.g., Pylayeva-Gupta, et al., Nat. Rev. Cancer, 2011, 11 (11), 761-774).
  • K-Ras and N-Ras substitutions are frequently observed in pancreatic (95% K-Ras), colon (47% K-Ras), lung (35% K-Ras) and melanoma (28% N-Ras).
  • Y 1 is N or CH
  • W and X are each independently selected from the group consisting of H, halo, and C1-4 alkyl;
  • each R B is H;
  • R B groups come together, in combination with the carbon atoms to which they are attached, to form a C3-6 carbocyclic group or a 3-6 membered heterocyclic group;
  • Cy 1 is selected from the group consisting of:
  • R 1 and R 5 are each independently selected from the group consisting of H, halo, OH, Ci-4 alkyl, and Ci -4 haloalkyl;
  • R 3 is selected from the group consisting of H and halo
  • R 2 and R 4 are each independently selected from the group consisting of H and halo;
  • R 6 is absent or selected from the group consisting of H and Ci-4 alkyl
  • At least one of W, X, R B , R 1 , R 2 , R 4 , R 5 , and R 6 is not H.
  • the compound is a compound of Formula la:
  • Y 1 is N.
  • A is selected from the group consisting of N, NH,
  • A is NH.
  • L is O.
  • W is selected from the group consisting of H, F, CI, Br, and CH 3 .
  • X is selected from the group consisting of H, F, CI, Br, and CH 3 . In some embodiments, X is F.
  • each R B is H. In some embodiments, two R B groups come together, in combination with the carbon atoms to which they are attached, to form a C3-6 carbocyclic group. In some embodiments, two R B groups come together, in combination with the carbon atoms to which they are attached, to form a C 3-6 carbocyclic group selected from:
  • two R groups come together, in combination with the carbon atoms to which they are attached, to form a phenyl group.
  • two R B groups come together, in combination with the carbon atoms to which they are attached, to form a 3-6 membered heterocyclic group.
  • two R B groups come together, in combination with the carbon atoms to which they are attached, to form a 3-6 membered heterocyclic group selected from:
  • R is selected from the group consisting of H, F, OH, and CF 3 .
  • R 2 is selected from the group consisting of H, F and CI.
  • R 3 is H or CI. In some embodiments, R 3 is H.
  • R 4 is selected from the group consisting of H and F.
  • R 5 is selected from the group consisting of H, F, OH, and CF 3 .
  • R 6 is absent. In some embodiments, R 6 is H or CH 3 . In some embodiments:
  • Y 1 is ;
  • W and X are each independently selected from the group consisting of H, halo, and CH 3 ;
  • each R B is H;
  • two R B groups come together, in combination with the carbon atoms to which they are attached, to form a C 3-6 carbocyclic group or a 3-6 membered heterocyclic group;
  • R 1 and R 5 are each independently selected from the group consisting of H, halo, OH, Ci-4 alkyl, and Ci -4 haloalkyl;
  • R 2 and R 4 are each independently selected from the group consisting of H and halo;
  • R 6 is absent or CH 3 .
  • Y 1 is ;
  • A is selected from the group consisting of N, NH, N(CH 3 ), S, O, and CFb;
  • L is selected from the group consisting of O, NH, S, and CEh;
  • W is selected from the group consisting of H, CI, Br, and CH3;
  • X is selected from the group consisting of H, F, CI, Br, and CH3;
  • each R B is H;
  • R B groups come together, in combination with the carbon atoms to which they are attached, to form a C3-6 carbocyclic group
  • R 1 and R 5 are each independently selected from the group consisting of H, halo, OH, Ci-4 alkyl, and C1-4 haloalkyl;
  • R 3 is H
  • R 2 and R 4 are each independently selected from the group consisting of H and halo;
  • R 6 is absent or CH 3 .
  • Y 1 is N
  • A is H
  • W is selected from the group consisting of H, CI, Br, and CH 3 ;
  • X is selected from the group consisting of H, F, CI, Br, and CH 3 ;
  • each R B is H;
  • R B groups come together, in combination with the carbon atoms to which they are attached, to form a C 3 -6 carbocyclic group
  • R 1 and R 5 are each independently selected from the group consisting of H, halo, OH, Ci-4 alkyl, and C1-4 haloalkyl;
  • R 3 is H
  • R 2 and R 4 are each independently selected from the group consisting of H and halo;
  • R 6 is absent.
  • the compound is a compound of Formula lb:
  • the compound is a compound of Formula Ic:
  • B is a C 3 -6 carbocyclic group or a 3-6 membered heterocyclic group.
  • the compound is a compound of Formula Id:
  • the compound is selected from the group consisting of:
  • the compound is 6,7-dimethoxy-N-(2-methyl-4- phenoxyphenyl)quinazolin-4-amine, or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is 6,7-dimethoxy-N-(2-methyl-4- phenoxyphenyl)quinazolin-4-amine 2,2,2-trifluoroacetate. In some embodiments, the compound is 6,7-dimethoxy-N-(2-methyl-4-phenoxyphenyl)quinazolin-4-amine hydrochloride.
  • Y 1 is N or CH
  • W and X are each independently selected from the group consisting of H, halo, and Ci -4 alkyl;
  • Cy 1 is selected from the group consisting of:
  • R 1 and R 5 are each independently selected from the group consisting of H, halo, OH, Ci-4 alkyl, and C1-4 haloalkyl; R 3 is selected from the group consisting of H and halo; and
  • R 2 and R 4 are each independently selected from the group consisting of H and halo; or
  • R 4 and R 5 come together, in combination with the carbon atoms to which they are attached, to form a C3-6 carbocyclic group or a 3-6 membered heterocyclic group;
  • R 6 is absent or selected from the group consisting of H and C alkyl.
  • the compound is a compound of Formula Ila:
  • Y 1 is N.
  • A is NH.
  • L is O.
  • W is selected from the group consisting of H, CI, Br, and CH .
  • X is selected from the group consisting of H, F, CI, Br, and CH 3 . In some embodiments, X is F.
  • R 1 is selected from the group consisting of H, F, OH, and CF3.
  • R 2 is selected from the group consisting of H and F.
  • R 3 is selected from the group consisting of H and CI. In some embodiments, R 3 is H.
  • R 4 is selected from the group consisting of H and F.
  • R 5 is selected from the group consisting of H, F, OH,
  • R 4 and R 5 are each H. In some embodiments, R 4 and R 5 come together, in combination with the carbon atoms to which they are attached, to form a C3-6 carbocyclic group. In some embodiments, R 4 and R 5 come together, in combination with the carbon atoms to which they are attached, to form a C3-6 carbocyclic group selected from:
  • R 4 and R 5 come together, in combination with the carbon atoms to which they are attached, to form a phenyl group. In some embodiments, R 4 and R 5 come together, in combination with the carbon atoms to which they are attached, to form 3-6 membered heterocyclic group. In some embodiments, R 4 and R 5 come together, in combination with the carbon atoms to which they are attached, to form 3-6 membered heterocyclic group selected from:
  • R 6 is absent. In some embodiments, R 6 is selected from the group consisting of H and CH 3 .
  • A is selected from the group consisting of N, NH, N(CH 3 ), S, O, and CH2;
  • L is selected from the group consisting of O, NH, S, and CH 2 ;
  • W and X are each independently selected from the group consisting of H, halo, and CH 3 ;
  • R 1 and R 5 are each independently selected from the group consisting of H, halo, OH, Ci-4 alkyl, and Ci -4 haloalkyl;
  • R 3 is selected from the group consisting of H and CI;
  • R 2 and R 4 are each independently selected from the group consisting of H and halo; or
  • R 4 and R 5 come together, in combination with the carbon atoms to which they are attached, to form a C 3-6 carbocyclic group or a 3-6 membered heterocyclic group;
  • R 6 is absent or CH 3 .
  • Y 1 is N
  • A is selected from the group consisting of N, NH, N(CH 3 ), S, O, and CH2;
  • L is selected from the group consisting of O, NH, S, and CH2;
  • W is selected from the group consisting of H, CI, Br, and CH 3 ;
  • X is selected from the group consisting of H, F, CI, Br, and CH 3 ;
  • R 1 and R 5 are each independently selected from the group consisting of H, halo, OH, Ci-4 alkyl, and C1-4 haloalkyl;
  • R 3 is selected from the group consisting of H and CI;
  • R 2 and R 4 are each independently selected from the group consisting of H and halo; or alternatively, R 4 and R 5 come together, in combination with the carbon atoms to which they are attached, to form a C3-6 carbocyclic group or a 3-6 membered heterocyclic group; and
  • R 6 is absent or CH 3 .
  • Y 1 is N
  • A is NH
  • W is selected from the group consisting of H, CI, Br, and CH3;
  • X is selected from the group consisting of H, F, CI, Br, and CH3;
  • R 1 and R 5 are each independently selected from the group consisting of H, halo, OH, Ci-4 alkyl, and C1-4 haloalkyl;
  • R 3 is selected from the group consisting of H and CI;
  • R 2 and R 4 are each independently selected from the group consisting of H and halo; or
  • R 4 and R 5 come together, in combination with the carbon atoms to which they are attached, to form a C 3 -6 carbocyclic group or a 3-6 membered heterocylic group;
  • R 6 is absent.
  • Y 1 is N
  • A is NH
  • W is selected from the group consisting of H, CI, Br, and CH3;
  • X is selected from the group consisting of H, F, CI, Br, and CH 3 ;
  • R 1 is selected from the group consisting of H, halo, OH, C1-4 alkyl, and C1-4 haloalkyl;
  • R 2 is selected from the group consisting of H and halo
  • R 3 is selected from the group consisting of H and CI;
  • R 4 and R 5 are each H;
  • R 6 is absent.
  • the compound is a compound of Formula lib:
  • the compound is a compound of Formula lie:
  • B is a C 3 - 6 carbocyclic group or a 3-6 membered heterocyclic group.
  • the compound is a compound of Formula lid:
  • the compound is selected from the rou consisting of:
  • L is selected from the group consisting of O, H, S, and Ci-3 alkylene
  • W and X are each independently selected from the group consisting of H, halo, and Ci -4 alkyl;
  • each R B is H;
  • R B groups come together, in combination with the carbon atoms to which they are attached, to form a C 3 -6 carbocyclic group
  • R 1 and R 5 are each independently selected from the group consisting of H, halo, OH, Ci-4 alkyl, and Ci -4 haloalkyl;
  • R 2 and R 4 are each independently selected from the group consisting of H and halo;
  • R 3 is selected from the group consisting of H and halo
  • R 6 is selected from the group consisting of H, C 1-3 alkyl, C3-6 cycloalkyl and C3-6 heterocycloalkyl.
  • L is selected from the group consisting of O, NH, S, and CH2. In some embodiments, L is 0.
  • W is selected from the group consisting of H, CI, Br, and CH 3 .
  • X is selected from the group consisting of H, F, CI, Br, and CH3. In some embodiments, X is F.
  • each R B is H. In some embodiments, two R B groups come together, in combination with the carbon atoms to which they are attached, to form a C3-6 carbocyclic group. In some embodiments, two R B groups come together, in combination with the carbon atoms to which they are attached, to form a phenyl group.
  • R 1 is selected from the group consisting of H, F, OH, and CF 3 .
  • R 2 is selected from the group consisting of H and F.
  • R 3 is selected from the group consisting of H and CI. In some embodiments, R 3 is H.
  • R 4 is selected from the group consisting of H and F.
  • R 5 is selected from the group consisting of H, F, OH, and CF 3 .
  • R 6 is selected from the group consisting of H, Ci -3 alkyl, and Cs- 6 cycloalkyl and 5-6 membered heterocycloalkyl. In some embodiments, R 6 is selected from the group consisting of H, CH 3 , cyclopentyl, 3-piperidinyl, 4- piperidinyl, and 3-pyrrolidinyl.
  • L is selected from the group consisting of O, NH, S, and CH 2 ;
  • W and X are each independently selected from the group consisting of H, halo, and CH 3 ;
  • each R B is H;
  • R B groups come together, in combination with the carbon atoms to which they are attached, to form a C 3- 6 carbocyclic group
  • R 1 and R 5 are each independently selected from the group consisting of H, halo, OH, Ci-4 alkyl, and Ci -4 haloalkyl;
  • R 2 and R 4 are each independently selected from the group consisting of H and halo;
  • R 3 is selected from the group consisting of H and CI.
  • R 6 is selected from the group consisting of H, Ci -3 alkyl, C 3- 6 cycloalkyl and 3- 6 membered heterocycloalkyl.
  • L is selected from the group consisting of O, NH, S, and CH_;
  • W is selected from the group consisting of H, CI, Br, and CH 3 ;
  • X is selected from the group consisting of H, F, CI, Br, and CH 3 ; each R B is H; or
  • R B groups come together, in combination with the carbon atoms to which they are attached, to form a C 3-6 carbocyclic group
  • R 1 and R 5 are each independently selected from the group consisting of H, halo, OH, Ci-4 alkyl, and Ci- 4 haloalkyl;
  • R 2 and R 4 are each independently selected from the group consisting of H and halo;
  • R 3 is selected from the group consisting of H and CI.
  • R 6 is selected from the group consisting of H, Ci -3 alkyl, C 5-6 cycloalkyl, and 5-6 membered heterocycloalkyl.
  • W is selected from the group consisting of H, CI, Br, and CH 3 ;
  • X is selected from the group consisting of H, F, CI, Br, and CH 3 ;
  • each R B is H;
  • R B groups come together, in combination with the carbon atoms to which they are attached, to form a C 3-6 carbocyclic group
  • R 1 and R 5 are each independently selected from the group consisting of H, halo, OH, Ci-4 alkyl, and Ci-4 haloalkyl;
  • R 2 and R 4 are each independently selected from the group consisting of H and halo;
  • R 3 is selected from the group consisting of H and CI.
  • R 6 is selected from the group consisting of H, CH 3 , C5-6 membered cycloalkyl, and 5-6 membered heterocycloalkyl.
  • the compound is a compound of Formula Ilia:
  • the compound is a compound of Formula Illb:
  • B is a C3-6 carbocyclic g a 3-6 membered heterocycloalkyl group.
  • the compound is a compound of Formula IIIc:
  • the compound is selected from the group consisting of:
  • the compound is 7-cyclopentyl-5-(4-phenoxyphenyl)- 7H-pyrrolo[2,3-d]pyrimidin-4-amine, or a pharmaceutically acceptable salt thereof.
  • the present application further provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound provided herein, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier.
  • the present application further provides a method of treating cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound provided herein.
  • the present application further provides a method of treating cancer in a patient in need thereof, comprising:
  • the cancer is selected from the group consisting of breast cancer, prostate cancer, esophageal cancer, colon cancer, endometrial cancer, brain cancer, bladder cancer, skin cancer, cancer of the uterus, cancer of the ovary, lung cancer, pancreatic cancer, renal cancer, prostate cancer, gastric cancer, stomach cancer, and hematological cancer.
  • the lung cancer is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, and lung carcinoid tumor.
  • the non-small cell lung cancer comprises an adenocarcinoma of the lung or squamous cell cancer of the lung.
  • the hematological cancer is selected from the group consisting of leukemia, lymphoma, and multiple myeloma.
  • the leukemia is selected from the group consisting of acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (AML), chronic myeloid leukemia (CML), hairy cell leukemia, T-cell prolymphocytic leukemia, juvenile myelomonocytic leukemia, and follicular lymphoma.
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia
  • AML acute myelogenous leukemia
  • CML chronic myeloid leukemia
  • hairy cell leukemia T-cell prolymphocytic leukemia
  • juvenile myelomonocytic leukemia juvenile myelomonocytic leukemia
  • follicular lymphoma follicular lymphoma
  • the lymphoma is Hodgkin's lymphoma or non- Hodgkin's lymphoma.
  • the method further comprises administering an additional therapeutic agent.
  • the additional therapeutic agent is a chemotherapeutic agent.
  • the additional therapeutic agent is a KSR inhibitor or a MEK inhibitor.
  • the additional therapeutic agent is a MEK inhibitor.
  • the MEK inhibitor is selected from the group consisting of trametinib, selumetinib, binimetinib, refametinib, pimasertib, cobimetinib, AZD8330, R04987655, R05126766, WX-554, E6201, MSC1936369B, PD-325901, CI-1040, RDEA119, CH5126766, GDC-0623, G-573, TAK-733, TAK- 133, CI-1 040/PD1 84352, AZD6244, PD318088, PD98059, PD334581, RDEAl 19, 6-methoxy-7-(3-morpholin-4-yl-propoxy)-4-(4-phenoxy-phenylamino)quinoline-3- carbonitrile, and 4-[3-Chloro-4-(l -methyl- lH-imidazol-2-ylsulfanyl)phenyla
  • the present application further provides a method of inhibiting a KSR in a cell, comprising contacting the cell with an effective amount of a compound provided herein, or a pharmaceutically acceptable salt thereof.
  • the present application further provides a method of inhibiting a KSR and a MEK in a cell, comprising contacting the cell with an effective amount of a compound provided herein, or a pharmaceutically acceptable salt thereof.
  • FIG. 3 shows labeling of ATP-binding pockets of purified KSR2:MEK1 complexes using an activity based probe (ATPbiotin).
  • FIG. 4 shows a graphical representation for ATPbiotin probe labeling of KSR2-MEK1 complexes in the presence of increasing free ATP as shown in FIG. 3. Corresponding ICso values are listed for both KSR2 and MEK1.
  • FIG. 5 shows results of a screen for direct competitors of ATP-biotin probe labeling of MEK and KSR.
  • FIG. 6 shows a crystal structure of KSR2:MEK1, assembled via a face-to-face interaction. Within this arrangement, two key phospho-regulatory residues in MEK1, Ser218 and Ser222, are buried and protected from the action of RAF. Inset includes a magnified view of interactions between KSR2, MEKl , and Compound 21.
  • Compounyd 21 and an extended segment that reinforces negative-regulatory interactions of KSR2 on MEKl are shown within 2
  • FIGs. 7A-7G shows results of a biolayer inferometry assay.
  • B-RAF and B- RAF mutants (F667E +/- R509H) were immobilized on sensor-heads and
  • KSR2:MEK1 or MEKl assembly was monitored. Curves indicate dose ranges of KSR2:MEK1 or MEKl from 625 nM to 10 ⁇ . In all plots, association occurred from 0 to 660 seconds, and dissociation was monitored thereafter up to 1500 seconds. Compound 21 and trametinib were added in the presence of KSR2:MEK1 at a concentration of 25 ⁇ each.
  • FIG. 8 shows phosphorylation of MEKl and KSR2:MEK1 with increasing amounts of B-RAF kinase over time. Reactions were initiated by the addition of 100 ⁇ ATP and halted at the indicated times. Each reaction was then immunoblotted and probed for pMEK and tMEK to detect RAF-mediated MEK phosphorylation.
  • FIGs. 9A-9C show phosphorylation of MEK1, KSR2:MEK1, and
  • KSR2 A690F :MEK1 by B-RAF over time in the presence of Compound 21 Relative pMEK was determined by quantification of band intensities from western blots using Image Lab Software 4.1.
  • FIGs. 10A-10B show KSR1 knockdown and Compound 21 treatment similarly block FBS-induced ERK activation.
  • HEK293 cells were serum starved for 24 hours and simultaneously treated with two independent siRNAs against KSR1.
  • siNTC was used as a scrambled control. After induction with 5% FBS, cells were harvested at 0, 30, 60, and 120 seconds.
  • FIG. 10B HEK293 cells were similarly serum starved and pre-treated with Compound 21 for 2 h before induction. The cells were then induced with FBS as in FIG. 10A.
  • FIG. 11 shows a western blot analysis of the treatment of cells with the Compound 21 in combination with a MEK inhibitor, trametinib.
  • the arrow indicates effective pathway knockdown by combination inhibitors.
  • FIG. 12 shows Compound 21 enhancing the efficacy of the clinical MEK inhibitor trametinib within cancer cell lines containing K-Ras mutations.
  • High BLISS scores indicate statistically significant synergy between Compound 21 and trametinib specifically within K-Ras mutant cells. Numbers listed within synergy matrices, representing % growth inhibition relative to DMSO controls, are mean of two replicates. Insets highlight dose responses of trametinib in the absence or presence of 1 ⁇ Compound 21.
  • FIGs. 13A-13D show data validating that Compound 21 is synergistic with trametinib in Ras-mutant cells.
  • FIG. 13A shows a BLISS score analysis of HCT-116, A549, A375, and SK-MEL-239 cells treated with Compound 21, sarcatinib, or lapatinib (range: 100-3000 in three-fold dilutions) in combination with trametinib (range: 0.01-100 in three-fold dilution).
  • BLISS scores were calculated based on combination assays as shown in Figure 4A.
  • FIG. 13B shows absolute BLISS score of the indicated drugs in combination with trametinib in Ras-mutant relative to RAF- mutant cell lines demonstrates selective synergy in Ras-mutant cell lines for
  • FIGs. 13C-13D show log of the combination index (CI) graphs of Compound 21 in combination with trametinib in HCT-116 vs. SK-MEL-239 cells as compared to the fractional effect (Fa). Negative CI over a broad Fa range within HCT-116, but not SK-MEL-239, indicates strong synergy.
  • FIGs. 14A-14B shows the increased potency of trametinib in the presence of Compound 21 within HCT-116 cells relative to the control cell line A375, which occurs through enhanced down-regulation of Ras-MAPK signaling (as measured by
  • FIGs. 15A-15C show the results of in vitro cell assays using Compound 21 and HTC-116 cells (FIG. 15A), A549 cells (FIG. 15B), and LOVO cells (FIG. 15C).
  • Combination drugs are increasingly recognized as a therapeutic modality for a variety of complex diseases including cancer (see, e.g., Glickman et al., Cell, 2012, 148(6), 1089-1098).
  • drug combinations may be required to improve treatment responses, minimize adverse events, or minimize development of resistance.
  • combination approaches have primarily focused on three strategies including co-targeting of a single pathway (see e.g., Chapman et al., Cancer Cell, 2014, 26(5), 603-604), different pathways (see e.g., Vora et al., Cancer Cell, 2014, 26(1), 136-149), or compensatory pathways (see e.g., Carver et al., Cancer Cell, 2011, 19(5), 575-586).
  • KSR Ras driven tumors
  • KSR controls Ras-dependent proliferation via direct interactions with several kinases in the MAPK cascade and also metabolism via AMP-activated protein kinase signaling (see e.g., Costanzo-Garvey et al., Cell Metab. 2009, 10(5), 366-378; Roy et al, Genes Dev. 2002, 16(4), 427-438; and Ritt et al., Methods Enzymol. 2006, 407, 224-237).
  • KSR belongs to a family of highly related kinases, including KSRl and KSR2, and the human RAF kinases (A-RAF, B-RAF, C-RAF). KSRl and KSR2 share 61% overall amino acid identity, and 71% amino acid identity between kinase domains. While both KSRl and KSR2 can interact with RAF, MEK, and ERK, KSRl has been shown to be prominently involved in MAPK signaling, while KSR2 was shown to impact cell growth through its interaction and functional impact on AMPK (see e.g., Fernandez et al., Mol. Cell. Biol. 2012, 32(18), 3718-3731).
  • KSRl knockout mice exhibit a rough hair phenotype
  • KSR2 knockout mice display a severe obese phenotype
  • Knockout of KSRl in RAS-driven tumor mouse models completely blocks tumorigenesis.
  • KSRl appears to be essential for RAS-driven tumorigenesis but not required for normal homeostasis (see e.g., Blasco et al., Cancer Cell, 2011, 19(5), 652-653).
  • KSR as a catalytically compromised kinase
  • pseudokinase a catalytically compromised kinase
  • Current models suggest that KSR functions as a scaffold to potentiate Ras signaling through the formation of macromolecular signaling complexes that include the Ras effector kinases RAF and MEK.
  • KSR likely forms a high affinity complex with inactive forms of MEK but once engaged by active RasGTP-RAF complexes, KSR adopts a distinct state where it can instead drive MEK
  • KSR antagonists Small molecules that could antagonize KSR dependent activities would be valuable tools that could be used to functionally annotate the pharmacology of this class of protein in Ras or RAF dependent cancers. Accordingly, the present application provides compounds that are useful as KSR antagonists and methods of using same. The following abbreviations may be used herein: calc. (calculated); d
  • Y 1 is N or CH
  • W and X are each independently selected from the group consisting of H, halo, and C1-4 alkyl; each R B is H; or
  • R B groups come together, in combination with the carbon atoms to which they are attached, to form a C 3 -6 carbocyclic group or a 3-6 membered heterocyclic group;
  • Cy 1 is selected from the group consisting of:
  • R 1 and R 5 are each independently selected from the group consisting of H, halo, OH, Ci-4 alkyl, and Ci-4 haloalkyl;
  • R 3 is selected from the group consisting of H and halo
  • R 2 and R 4 are each independently selected from the group consisting of H and halo;
  • R 6 is absent or selected from the group consisting of H and Ci-4 alkyl.
  • At least one of W, X, R B , R 1 , R 2 , R 4 , R 5 , and R 6 is not H.
  • Cy 1 is selected from the group consisting of:
  • the compound of Formula I is a compound of Formula
  • Y 1 is N. In some embodiments, Y 1 is CH.
  • A is selected from the group consisting of N, NH, N(CH 3 ), S, O, and CH 2 .
  • A is NH.
  • A is N(CH 3 ).
  • A is S.
  • L is selected from the group consisting of O, NH, S, and CH 2 . In some embodiments, L is O. In some
  • L is NH. In some embodiments, L is S. In some embodiments, L is CH2. In some embodiments, L is CH(OH). In some embodiments, L is CH(NH 2 ). In some embodiments, L is selected from the group consisting of CH(OH), CH(NH 2 ), CH(NHCH3), CH(NHCH 2 CH 3 ), CH(NHCH 2 CH 2 CH 3 ), CH(NHCH(CH 3 ) 2 ),
  • W is selected from the group consisting of H, halo, and CH 3 . In some embodiments, W is selected from the group consisting of H, F, CI, Br, and Ci-4 alkyl. In some embodiments, W is selected from the group consisting of H, F, CI, Br, and CH 3 . In some embodiments, W is selected from the group consisting of H, CI, Br, and CH 3 . In some embodiments, W is H. In some embodiments, W is halo. In some embodiments, W is CI or Br. In some embodiments, W is C1-4 alkyl. In some embodiments, W is CH 3 .
  • X is selected from the group consisting of H, halo, and
  • X is selected from the group consisting of H, F, CI, Br, and Ci-4 alkyl. In some embodiments, X is selected from the group consisting of H, F, CI, Br, and CH 3 . In some embodiments, X is H. In some embodiments, X is halo. In some embodiments, X is selected from the group consisting of F, CI, and Br. In some embodiments, X is F. In some embodiments, X is Ci-4 alkyl. In some embodiments, X is CH 3 .
  • each B is H.
  • two R B groups come together, in combination with the carbon atoms to which they are attached, to form a C 3-6 carbocyclic group.
  • two R B groups come together, in combination with the carbon atoms to which they are attached, to form a C3-6 carbocyclic group selected from:
  • two R B groups come together, in combination with the carbon atoms to which they are attached, to form a phenyl group.
  • two R B groups come together, in combination with the carbon atoms to which they are attached, to form a 3-6 membered heterocyclic group. In some embodiments, two R B groups come together, in combination with the carbon atoms to which they are attached, to form a 3-6 membered heterocyclic group selected from:
  • R is selected from the group consisting of H, halo, OH, and Ci-4haloalkyl.
  • R 1 is selected from the group consisting of H, halo, OH, and CF 3 .
  • R 1 is selected from the group consisting of H, F, CI, Br, OH, and Ci-4haloalkyl.
  • R 1 is selected from the group consisting of H, F, CI, Br, OH, and CF 3 .
  • R 1 is selected from the group consisting of H, F, OH, and CF 3 .
  • R 2 is selected from the group consisting of H, F, CI, and Br. In some embodiments, R 2 is selected from the group consisting of H, F, and CI. In some embodiments, R 2 is selected from the group consisting of H and F. In some embodiments, R 2 is H. In some embodiments, R 2 is F.
  • R 3 is H. In some embodiments, R 3 is halo. In some embodiments, R 3 is selected from the group consisting of F, CI, and Br. In some embodiments, R 3 is CI. In some embodiments, R 3 is selected from the group consisting of H and CI.
  • R 4 is selected from the group consisting of H, F, CI, and Br. In some embodiments, R 4 is selected from the group consisting of H and F. In some embodiments, R 4 is H. In some embodiments, R 4 is F.
  • R 5 is selected from the group consisting of H, halo, OH, and Ci-4 haloalkyl. In some embodiments, R 5 is selected from the group consisting of H, halo, OH, and CF 3 . In some embodiments, R 5 is selected from the group consisting of H, F, CI, Br, OH, and Cwhaloalkyl. In some embodiments, R 5 is selected from the group consisting of H, F, CI, Br, OH, and CF 3 . In some embodiments,
  • R 5 is selected from the group consisting of H, F, OH, and CF 3 . In some embodiments, R 5 is H. In some embodiments, R 5 is F. In some embodiments, R 5 is OH. In some embodiments, R 5 is CF 3 .
  • R 6 is absent. In some embodiments, R 6 is selected from the group consisting of H and Ci-4 alkyl. In some embodiments, R 6 is selected from the group consisting of H and CH 3 . In some embodiments, R 6 is H. In some embodiments, R 6 is CH 3 .
  • A is selected from the group consisting of N, NH, N(CH 3 ), S, O, and CBb;
  • L is selected from the group consisting of O, NH, S, and CH 2 ;
  • W and X are each independently selected from the group consisting of H, halo, and CH 3 ;
  • each R B is H;
  • R B groups come together, in combination with the carbon atoms to which they are attached, to form a C 3-6 carbocyclic group
  • R 1 and R 5 are each independently selected from the group consisting of H, halo, OH, Ci-4 alkyl, and Ci-4 haloalkyl;
  • R 3 is selected from the group consisting of H and CI;
  • R 2 and R 4 are each independently selected from the group consisting of H and halo;
  • R 6 is absent or CH 3 .
  • Y 1 is N
  • A is selected from the group consisting of N, NH, N(CH 3 ), S, O, and CH 2 ;
  • L is selected from the group consisting of O, NH, S, and CEb;
  • W is selected from the group consisting of H, CI, Br, and CH 3 ;
  • X is selected from the group consisting of H, F, CI, Br, and CH 3 ;
  • each R B is H;
  • R B groups come together, in combination with the carbon atoms to which they are attached, to form a C 3 - 6 carbocyclic group
  • R 1 and R 5 are each independently selected from the group consisting of H, halo, OH, Ci-4 alkyl, and Ci-4 haloalkyl;
  • R 3 is selected from the group consisting of H and CI;
  • R 2 and R 4 are each independently selected from the group consisting of H and halo;
  • R 6 is absent or CH 3 .
  • Y 1 is N
  • A is NH
  • W is selected from the group consisting of H, CI, Br, and CH 3 ;
  • X is selected from the group consisting of H, F, CI, Br, and CH 3 ;
  • each R B is H;
  • R B groups come together, in combination with the carbon atoms to which they are attached, to form a C 3 - 6 carbocyclic group
  • R 1 and R 5 are each independently selected from the group consisting of H, halo, OH, Ci-4 alkyl, and Ci-4 haloalkyl;
  • R 3 is selected from the group consisting of H and CI; and R 2 and R 4 are each independently selected from the group consisting of H and halo; and
  • R 6 is absent.
  • the compound of Formula I is a compound of Formula la:
  • Y 1 is or CH
  • A is selected from the group consisting of N, NH, N(Ci- 4 alkyl), S, O, and Ci -3 alkylene;
  • L is selected from the group consisting of O, NH, S, and Ci -3 alkylene;
  • W and X are each independently selected from the group consisting of H, halo, and Ci-4 alkyl;
  • each R B is H;
  • R B groups come together, in combination with the carbon atoms to which they are attached, to form a C 3-6 carbocyclic group
  • R 1 and R 5 are each independently selected from the group consisting of H, halo, OH, Ci-4 alkyl, and Ci-4 haloalkyl;
  • R 3 is selected from the group consisting of H and halo
  • R 2 and R 4 are each independently selected from the group consisting of H and halo;
  • R 6 is absent or selected from the group consisting of H and Ci-4 alkyl.
  • the compound of Formula I is a compound of Formula lb:
  • the compound of Formula I is a compound of Formula
  • B is a C3-6 carbocyclic group or a 3-6 membered heterocyclic group
  • A, W, X, L, R 1 , R 2 , R 3 , R 4 , and R 5 are as defined above for compounds of Formula I.
  • the compound of Formula I is a compound of Formula
  • the compound of Formula I is a pharmaceutically acceptable salt.
  • the compound of Formula I is a hydrochloride (i.e. HCl) salt.
  • the compound of Formula I is a 2,2,2,- trifluoroacetic acid (i.e., TFA) salt.
  • the compound is 6,7-dimethoxy-N-(2-methyl-4- phenoxyphenyl)quinazolin-4-amine (21), or a pharmaceutically acceptable salt thereof.
  • the compound is 6,7-dimethoxy-N-(2-methyl-4- phenoxyphenyl)quinazolin-4-amine 2,2,2-trifluoroacetate (21 -TFA).
  • the compound is 6,7-dimethoxy-N-(2-methyl-4- phenoxyphenyl)quinazolin-4-amine hydrochloride (21 ⁇ ).
  • Y 1 is N or CH
  • W and X are each independently selected from the group consisting of H, halo, and Ci-4 alkyl;
  • Cy 1 is selected from the group consisting of:
  • R 1 and R 5 are each independently selected from the group consisting of H, halo, OH, Ci-4 alkyl, and Ci-4 haloalkyl;
  • R 3 is selected from the group consisting of H and halo
  • R 2 and R 4 are each independently selected from the group consisting of H and halo; or
  • R 4 and R 5 come together, in combination with the carbon atoms to which they are attached, to form a C3-6 carbocyclic group or a 3-6 membered heterocyclic group;
  • R 6 is absent or selected from the group consisting of H and C1-4 alkyl.
  • the compound of Formula II is a compound of Formula
  • Y 1 is N. In some embodiments, Y 1 is CH.
  • A is selected from the group consisting of N, NH, N(CH 3 ), S, O, and CH2.
  • A is NH.
  • A is N(CH 3 ).
  • A is S.
  • A is O. In some embodiments, A is CH2. In some embodiments, A is selected from the group consisting of CH(OH), CH(NH 2 ), CH(NHCH 3 ),
  • CH(NHCH 2 CH 3 ), CH(NHCH 2 CH2CH 3 ), CH(NHCH(CH 3 ) 2 ), CH(NHCH 2 CH(CH 3 )2), CH(NHC(CH 3 ) 3 ), and CH(NHCH 2 CH CH 2 ).
  • L is selected from the group consisting of O, NH, S, and CH 2 . In some embodiments, L is O. In some
  • W is selected from the group consisting of H, halo, and CH 3 . In some embodiments, W is selected from the group consisting of H, F, CI, Br, and Ci-4 alkyl. In some embodiments, W is selected from the group consisting of H, F, CI, Br, and CH3. In some embodiments, W is selected from the group consisting of H, CI, Br, and CH3. In some embodiments, W is H. In some embodiments, W is halo. In some embodiments, W is CI or Br. In some embodiments, W is C1-4 alkyl. In some embodiments, W is CH3.
  • X is selected from the group consisting of H, halo, and
  • X is selected from the group consisting of H, F, CI, Br, and Ci-4 alkyl. In some embodiments, X is selected from the group consisting of H, F, CI, Br, and CH 3 . In some embodiments, X is H. In some embodiments, X is halo. In some embodiments, X is selected from the group consisting of F, CI, and Br. In some embodiments, X is F. In some embodiments, X is Ci-4 alkyl. In some embodiments, X is CH3.
  • R 1 is selected from the group consisting of H, halo, OH, and Ci- 4 haloalkyl. In some embodiments, R 1 is selected from the group consisting of H, halo, OH, and CF3. In some embodiments, R 1 is selected from the group consisting of H, F, CI, Br, OH, and Ci-4 haloalkyl. In some embodiments, R 1 is selected from the group consisting of H, F, CI, Br, OH, and CF3. In some embodiments,
  • R 1 is selected from the group consisting of H, F, OH, and CF 3 .
  • R 2 is selected from the group consisting of H, F, CI, and Br. In some embodiments, R 2 is selected from the group consisting of H, F, and CI. In some embodiments, R 2 is selected from the group consisting of H and F. In some embodiments, R 2 is H. In some embodiments, R 2 is F.
  • R 3 is H. In some embodiments, R 3 is halo. In some embodiments, R 3 is selected from the group consisting of F, CI, and Br. In some embodiments, R 3 is CI. In some embodiments, R 3 is selected from the group consisting of H and CI.
  • R 4 is selected from the group consisting of H, F, CI, and Br. In some embodiments, R 4 is selected from the group consisting of H, F, and CI. In some embodiments, R 4 is selected from the group consisting of H and F. In some embodiments, R 4 is H. In some embodiments, R 4 is F.
  • R 5 is selected from the group consisting of H, halo,
  • R 5 is selected from the group consisting of H, halo, OH, and CF3. In some embodiments, R 5 is selected from the group consisting of H, F, CI, Br, OH, and Ci -4 haloalkyl. In some embodiments, R 5 is selected from the group consisting of H, F, CI, Br, OH, and CF3. In some embodiments,
  • R 5 is selected from the group consisting of H, F, OH, and CF3. In some embodiments, R 5 is H. In some embodiments, R 5 is F. In some embodiments, R 5 is OH. In some embodiments, R 5 is CF3.
  • R 4 and R 5 are each H. In some embodiments, R 4 and R 5 come together, in combination with the carbon atoms to which they are attached, to form a C3-6 carbocyclic group. In some embodiments, R 4 and R 5 come together, in combination with the carbon atoms to which they are attached, to form a C3-6 carbocyclic group selected from:
  • R 4 and R 5 come together, in combination with the carbon atoms to which they are attached, to form a phenyl group.
  • R 4 and R 5 come together, in combination with the carbon atoms to which they are attached, to form 3-6 membered heterocyclic group. In some embodiments, R 4 and R 5 come together, in combination with the carbon atoms to which they are attached, to form 5-6 membered heteroaryl group. In some embodiments, R 4 and R 5 come together, in combination with the carbon atoms to which they are attached, to form 3-6 membered heterocycloalkyl group. In some embodiments, R 4 and R 5 come together, in combination with the carbon atoms to which they are ected from:
  • R 6 is absent. In some embodiments, R 6 is selected from the group consisting of H and Ci-4 alkyl. In some embodiments, R 6 is selected from the group consisting of H and CH 3 . In some embodiments, R 6 is H. In some embodiments, R 6 is CH3.
  • Y 1 is N
  • A is selected from the group consisting of N, H, N(CH 3 ), S, O, and CH2;
  • L is selected from the group consisting of O, NH, S, and CH2;
  • W and X are each independently selected from the group consisting of H, halo, and CH3;
  • R 1 and R 5 are each independently selected from the group consisting of H, halo, OH, Ci-4 alkyl, and C1-4 haloalkyl;
  • R 3 is selected from the group consisting of H and CI.
  • R 2 and R 4 are each independently selected from the group consisting of H and halo; or
  • R 4 and R 5 come together, in combination with the carbon atoms to which they are attached, to form a C3-6 carbocyclic group
  • R 6 is absent or CH 3 .
  • Y 1 is N
  • A is selected from the group consisting of N, NH, N(CH 3 ), S, O, and CHb;
  • L is selected from the group consisting of O, NH, S, and CH2;
  • W is selected from the group consisting of H, CI, Br, and CH 3 ;
  • X is selected from the group consisting of H, F, CI, Br, and CH 3 ;
  • R 1 and R 5 are each independently selected from the group consisting of H, halo, OH, Ci-4 alkyl, and Ci-4 haloalkyl;
  • R 3 is selected from the group consisting of H and CI.
  • R 2 and R 4 are each independently selected from the group consisting of H and halo; or
  • R 4 and R 5 come together, in combination with the carbon atoms to which they are attached, to form a C3-6 carbocyclic group
  • R 6 is absent or CH3.
  • A is H
  • W is selected from the group consisting of H, CI, Br, and CH 3 ;
  • X is selected from the group consisting of H, F, CI, Br, and CH 3 ;
  • R 1 and R 5 are each independently selected from the group consisting of H, halo, OH, Ci-4 alkyl, and C1-4 haloalkyl;
  • R 3 is selected from the group consisting of H and CI.
  • R 2 and R 4 are each independently selected from the group consisting of H and halo; or
  • R 4 and R 5 come together, in combination with the carbon atoms to which they are attached, to form a C3-6 carbocyclic group
  • R 6 is absent.
  • Y 1 is N
  • A is H
  • W is selected from the group consisting of H, CI, Br, and CH3;
  • X is selected from the group consisting of H, F, CI, Br, and CH3;
  • R 1 is selected from the group consisting of H, halo, OH, Ci-4 alkyl, and C1-4 haloalkyl;
  • R 2 is selected from the group consisting of H and halo
  • R 3 is selected from the group consisting of H and CI; R 4 and R 5 are each H; and
  • R 6 is absent.
  • the compound of Formula II is a compound of Formula
  • Y 1 is N or CH
  • Y 2 is selected from the group consisting of N, NH, and N(Ci-4 alkyl);
  • A is selected from the group consisting of N, NH, N(Ci-4 alkyl), S, O, and C 1-3 alkylene;
  • L is selected from the group consisting of O, NH, S, and Ci -3 alkylene;
  • W and X are each independently selected from the group consisting of H, halo, and Ci -4 alkyl;
  • R 1 and R 5 are each independently selected from the group consisting of H, halo, OH, Ci-4 alkyl, and Ci -4 haloalkyl;
  • R 3 is selected from the group consisting of H and halo
  • R 2 and R 4 are each independently selected from the group consisting of H and halo; or
  • R 4 and R 5 come together, in combination with the carbon atoms to which they are attached, to form a C3-6 carbocyclic group.
  • the compound of Formula II is a compound of Formula lib:
  • the compound of Formula II is a compound of Formula lie:
  • B is a C 3 - 6 carbocyclic group or a 3-6 membered heterocyclic group
  • A, W, X, L, R 1 , R 2 , and R 3 are as defined above for compounds of Formula II.
  • B is a C3-6 carbocyclic group.
  • B is a 3-6 membered heterocyclic group.
  • the compound of Formula II is a compound of Formula lid:
  • the compound of Formula II is selected from the group consisting of:
  • the compound of Formula II is a pharmaceutically acceptable salt.
  • the compound of Formula II is a hydrochloride (i.e. HC1) salt.
  • the compound of Formula II is a 2,2,2,- trifluoroacetic acid (i.e., TFA) salt.
  • L is selected from the group consisting of O, NH, S, and Ci -3 alkylene;
  • W and X are each independently selected from the group consisting of H, halo, and Ci-4 alkyl;
  • each R B is H;
  • R B groups come together, in combination with the carbon atoms to which they are attached, to form a C3-6 carbocyclic group
  • R 1 and R 5 are each independently selected from the group consisting of H, halo, OH, Ci-4 alkyl, and C1-4 haloalkyl;
  • R 2 and R 4 are each independently selected from the group consisting of H and halo;
  • R 3 is selected from the group consisting of H and halo
  • R 6 is selected from the group consisting of H, C1-3 all y 1, C 3 -6 cycloalkyl and 3- 6 membered heterocycloalkyl.
  • L is selected from the group consisting of O, NH, S, and CH2. In some embodiments, L is O.
  • W is selected from the group consisting of H, halo, and CH3. In some embodiments, W is selected from the group consisting of H, F, CI, Br, and Ci-4 alkyl. In some embodiments, W is selected from the group consisting of H, F, CI, Br, and CH3. In some embodiments, W is selected from the group consisting of H, CI, Br, and CH3. In some embodiments, W is H. In some embodiments, W is halo. In some embodiments, W is CI or Br. In some embodiments, W is C1-4 alkyl. In some embodiments, W is CH3.
  • X is selected from the group consisting of H, halo, and CH3. In some embodiments, X is selected from the group consisting of H, F, CI, Br, and Ci-4 alkyl. In some embodiments, X is selected from the group consisting of H, F, CI, Br, and CH 3 . In some embodiments, X is H. In some embodiments, X is halo. In some embodiments, X is selected from the group consisting of F, CI, and Br. In some embodiments, X is F. In some embodiments, X is C1-4 alkyl. In some embodiments, X is CH 3 .
  • each R B is H. In some embodiments, two R B groups come together, in combination with the carbon atoms to which they are attached, to form a C 3-6 carbocyclic group. In some embodiments, two R B groups come together, in combination with the carbon atoms to which they are attached, to form a phenyl group.
  • R 1 is selected from the group consisting of H, halo,
  • R 1 is selected from the group consisting of H, halo, OH, and CF 3 . In some embodiments, R 1 is selected from the group consisting of H, F, CI, Br, OH, and Ci- 4 haloalkyl. In some embodiments, R 1 is selected from the group consisting of H, F, CI, Br, OH, and CF 3 . In some embodiments,
  • R 1 is selected from the group consisting of H, F, OH, and CF 3 .
  • R 2 is selected from the group consisting of H, F, CI, and Br. In some embodiments, R 2 is selected from the group consisting of H and F. In some embodiments, R 2 is H. In some embodiments, R 2 is F.
  • R 3 is H. In some embodiments, R 3 is halo. In some embodiments, R 3 is selected from the group consisting of F, CI, and Br. In some embodiments, R 3 is CI. In some embodiments, R 3 is selected from the group consisting of H and CI.
  • R 4 is selected from the group consisting of H, F, CI, and Br. In some embodiments, R 4 is selected from the group consisting of H and F. In some embodiments, R 4 is H. In some embodiments, R 4 is F.
  • R 5 is selected from the group consisting of H, halo, OH, and Ci -4 haloalkyl. In some embodiments, R 5 is selected from the group consisting of H, halo, OH, and CF3. In some embodiments, R 5 is selected from the group consisting of H, F, CI, Br, OH, and Ci -4 haloalkyl. In some embodiments, R 5 is selected from the group consisting of H, F, CI, Br, OH, and CF3. In some embodiments,
  • R 5 is selected from the group consisting of H, F, OH, and CF3. In some embodiments, R 5 is H. In some embodiments, R 5 is F. In some embodiments, R 5 is OH. In some embodiments, R 5 is CF3.
  • R 6 is selected from the group consisting of H, Ci-3 alkyl, and C3-6 cycloalkyl. In some embodiments, R 6 is selected from the group consisting of H, Ci-3 alkyl, and C5-6 cycloalkyl. In some embodiments, R 6 is selected from the group consisting of H, Ci -3 alkyl, and cyclopentyl. In some embodiments, R 6 is selected from the group consisting of H, CH3, and C 5-6 cycloalkyl. In some embodiments, R 6 is selected from the group consisting of H, CH 3 , and cyclopentyl.
  • R 6 is selected from the group consisting of H, C1-3 alkyl, and C 5 -6 cycloalkyl and 5-6 membered heterocycloalkyl. In some embodiments, R 6 is selected from the group consisting of H, CH3, cyclopentyl, 3-piperidinyl, 4-piperidinyl, and 3- pyrrolidinyl.
  • L is selected from the group consisting of O, NH, S, and CH2;
  • W and X are each independently selected from the group consisting of H, halo, and CH 3 ;
  • each R B is H;
  • R B groups come together, in combination with the carbon atoms to which they are attached, to form a C3-6 carbocyclic group
  • R 1 and R 5 are each independently selected from the group consisting of H, halo, OH, Ci-4 alkyl, and Ci -4 haloalkyl;
  • R 2 and R 4 are each independently selected from the group consisting of H and halo;
  • R 3 is selected from the group consisting of H and CI.
  • R 6 is selected from the group consisting of H, Ci-3 alkyl, and C3-6 cycloalkyl.
  • L is selected from the group consisting of O, NH, S, and CBb;
  • W is selected from the group consisting of H, CI, Br, and CH 3 ;
  • X is selected from the group consisting of H, F, CI, Br, and CH3;
  • each R B is H;
  • R B groups come together, in combination with the carbon atoms to which they are attached, to form a C3-6 carbocyclic group
  • R 1 and R 5 are each independently selected from the group consisting of H, halo, OH, Ci-4 alkyl, and Ci-4 haloalkyl;
  • R 2 and R 4 are each independently selected from the group consisting of H and halo;
  • R 3 is selected from the group consisting of H and CI.
  • R 6 is selected from the group consisting of H, C1-3 alkyl, and C5-6 cycloalkyl.
  • W is selected from the group consisting of H, CI, Br, and CH 3 ;
  • X is selected from the group consisting of H, F, CI, Br, and CH 3 ;
  • each R B is H;
  • R B groups come together, in combination with the carbon atoms to which they are attached, to form a C3-6 carbocyclic group
  • R 1 and R 5 are each independently selected from the group consisting of H, halo, OH, Ci-4 alkyl, and Ci- 4 haloalkyl;
  • R 2 and R 4 are each independently selected from the group consisting of H and halo;
  • R 3 is selected from the group consisting of H and CI.
  • R 6 is selected from the group consisting of H, CH 3 , and C5-6 membered cycloalkyl.
  • the compound of Formula III is a compound of Formula Ilia:
  • the compound of Formula III is a compound of Formula Illb:
  • B is a C 3-6 carbocyclic group or a 3-6 membered heterocyclic group
  • W, X, R 1 , R 2 , R 3 , and R 6 are as defined above for compounds of Formula III.
  • B is a C 3-6 carbocyclic group.
  • B is a 3-6 membered heterocyclic group.
  • the compound of Formula III is a compound of Formula IIIc:
  • the compound of Formula III is a pharmaceutically acceptable salt.
  • the compound of Formula III is a hydrochloride (i.e. HC1) salt.
  • the compound of Formula III is a 2,2,2,-trifluoroacetic acid (i.e., TFA) salt.
  • the compound of Formula III is selected from the group consisting of:
  • the compound is 7-cyclopentyl-5-(4-phenoxyphenyl)- 7H-pyrrolo[2,3-d]pyrimidin-4-amine (94), or a pharmaceutically acceptable salt thereof.
  • the present application further provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound provided herein (e.g., a compound of Formula I, a compound of Formula II, a compound of Formula III, or a compound selected from group the consisting of compounds (1)-(114)) or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier.
  • the present invention also includes pharmaceutically acceptable salts of the compounds described herein.
  • pharmaceutically acceptable salts refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form.
  • examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like.
  • the pharmaceutically acceptable salts of the present invention include the non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
  • the pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods.
  • such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, non-aqueous media like ether, ethyl acetate, alcohols (e.g., methanol, ethanol, iso-propanol, or butanol) or acetonitrile (ACN) are preferred.
  • non-aqueous media like ether, ethyl acetate, alcohols (e.g., methanol, ethanol, iso-propanol, or butanol) or acetonitrile (ACN) are preferred.
  • suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977), each of which is incorporated herein by reference in its entirety.
  • the compounds described herein include
  • the compounds provided herein can be administered in the form of pharmaceutical compositions; thus, the methods described herein can include administering pharmaceutical compositions provided herein.
  • the present application provides a pharmaceutical composition comprising a compound provided herein (e.g., a compound of Formula I, a compound of Formula II, a compound of Formula III or a compound selected from group the consisting of compounds (1)-(114)), or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier.
  • compositions can be prepared as described herein or elsewhere, and can be administered by a variety of routes, depending upon whether local or systemic treatment is desired and upon the area to be treated.
  • Administration may be pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal or intranasal), oral, or parenteral.
  • Parenteral administration may include, but is not limited to intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular injection or infusion; or intracranial, (e.g., intrathecal, intraocular, or intraventricular) administration.
  • Parenteral administration can be in the form of a single bolus dose, or may be, for example, by a continuous perfusion pump.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • the active ingredient is typically mixed with an excipient, diluted by an excipient or enclosed within such a carrier in the form of, for example, a capsule, sachet, paper, or other container.
  • an excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient.
  • compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.
  • excipients include, without limitation, lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose,
  • the formulations can additionally include, without limitation, lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates; sweetening agents; flavoring agents, or combinations thereof.
  • lubricating agents such as talc, magnesium stearate, and mineral oil
  • wetting agents such as talc, magnesium stearate, and mineral oil
  • emulsifying and suspending agents such as methyl- and propylhydroxy-benzoates
  • sweetening agents such as methyl- and propylhydroxy-benzoates
  • the active compounds can be effective over a wide dosage range and are generally administered in a therapeutically effective amount. It will be understood, however, that the amount of the compound actually administered and the schedule of administration will usually be determined by a physician, according to the relevant circumstances, including the condition to be treated, the chosen route of
  • the compounds provided herein, including salts thereof, can be prepared using known organic synthesis techniques and can be synthesized according to any of numerous possible synthetic routes.
  • the reactions for preparing compounds described herein can be carried out in suitable solvents which can be readily selected by one of skill in the art of organic synthesis.
  • suitable solvents can be substantially non-reactive with the starting materials (reactants), the intermediates, or products at the temperatures at which the reactions are carried out, (e.g., temperatures which can range from the solvent's freezing temperature to the solvent's boiling temperature).
  • a given reaction can be carried out in one solvent or a mixture of more than one solvent.
  • suitable solvents for a particular reaction step can be selected by the skilled artisan.
  • Preparation of compounds described herein can involve the protection and deprotection of various chemical groups.
  • the need for protection and deprotection, and the selection of appropriate protecting groups, can be readily determined by one skilled in the art.
  • the chemistry of protecting groups can be found, for example, in T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3 rd Ed., Wiley & Sons, Inc., New York (1999).
  • Reactions can be monitored according to any suitable method known in the art.
  • product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 3 ⁇ 4 or 13 C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), mass spectrometry, or by chromatographic methods such as high performance liquid chromatography (HPLC), liquid chromatography-mass spectroscopy (LCMS), or thin layer chromatography (TLC).
  • HPLC high performance liquid chromatography
  • LCMS liquid chromatography-mass spectroscopy
  • TLC thin layer chromatography
  • Compounds can be purified by those skilled in the art by a variety of methods, including high performance liquid chromatography (FfPLC) and normal phase silica chromatography.
  • FfPLC high performance liquid chromatography
  • FfPLC normal phase silica chromatography
  • each divalent linking substituent include both the forward and backward forms of the linking substituent.
  • - NR(CR'R")n- includes both -NR(CR'R") n - and -(CR'R") n NR-.
  • the Markush variables listed for that group are understood to be linking groups.
  • n-membered where n is an integer typically describes the number of ring-forming atoms in a moiety where the number of ring-forming atoms is n.
  • piperidinyl is an example of a 6-membered heterocycloalkyl ring
  • pyrazolyl is an example of a 5-membered heteroaryl ring
  • pyridyl is an example of a 6- membered heteroaryl ring
  • 1,2,3,4-tetrahydro-naphthalene is an example of a 10- membered cycloalkyl group.
  • the phrase "optionally substituted” means unsubstituted or substituted.
  • substituted means that a hydrogen atom is removed and replaced by a substituent. It is to be understood that substitution at a given atom is limited by valency. For example, where one or more bonds are shown as optionally being a single or double bond ⁇ i.e., ) ; it is understood that the bonds comply with standard valency rules.
  • Cn-m indicates a range which includes the endpoints, wherein n and m are integers and indicate the number of carbons. Examples include Ci-4, Ci-6, and the like.
  • C n -m alkyl employed alone or in combination with other terms, refers to a saturated hydrocarbon group that may be straight-chain or branched, having n to m carbons.
  • alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, R-propyl, isopropyl, «-butyl, tert- butyl, isobutyl, sec-butyl; higher homologs such as 2 -methyl- 1 -butyl, n-pentyl, 3- pentyl, w-hexyl, 1,2,2-trimethylpropyl, and the like.
  • the alkyl group contains from 1 to 6 carbon atoms, from 1 to 4 carbon atoms, from 1 to 3 carbon atoms, or 1 to 2 carbon atoms.
  • C n -m alkenyl refers to an alkyl group having one or more double carbon-carbon bonds and having n to m carbons.
  • Example alkenyl groups include, but are not limited to, ethenyl, «-propenyl, isopropenyl, rc-butenyl, sec- butenyl, and the like.
  • the alkenyl moiety contains 2 to 4 carbon atoms.
  • C n -m alkylene employed alone or in combination with other terms, refers to a divalent alkyl linking group having n to m carbons.
  • alkylene groups include, but are not limited to, ethan-l,2-diyl, propan- 1,3-diyl, propan-l,2-diyl, butan-l,4-diyl, butan-l,3-diyl, butan-l,2-diyl, 2-methyl- propan-l,3-diyl, and the like.
  • the alkylene moiety contains 2 to 6, 2 to 4, 2 to 3, 1 to 6, 1 to 4, or 1 to 2 carbon atoms.
  • Carbocycle refers to an aromatic (e.g., phenyl) or non-aromatic cyclic hydrocarbon including cyclized alkyl and/or alkenyl groups.
  • Carbocycles include, for example, 3-6 ring forming carbon atoms (i.e., C 3 -6
  • carbocycle can be mono- or polycyclic (e.g., having two or more fused rings).
  • the carbocycle is a C5-6 carbocycle.
  • Example carbocycles include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and phenyl.
  • halo refers to F, CI, Br, or I. In some embodiments, a halo is F, CI, or Br. In some embodiments, a halo is F.
  • Cn-mhaloalkyl refers to an alkyl group having from one halogen atom to 2s+l halogen atoms which may be the same or different, where "s" is the number of carbon atoms in the alkyl group, wherein the alkyl group has n to m carbon atoms.
  • the haloalkyl group is fluorinated only.
  • the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.
  • heterocycle refers to a heteroaryl or heterocycloalkyl group having at least one heteroatom ring member selected from O, N, and S.
  • the heterocycle is a 3-6 membered heterocycle.
  • the heterocycle is a 5-6 membered heterocycle.
  • the heterocycle is a heteroaryl.
  • the heterocycle is a heterocycloalkyl.
  • heteroaryl refers to an aromatic heterocycle having at least one heteroatom ring member selected from O, N, and S.
  • the heteroaryl ring has 1, 2, 3, or 4 heteroatom ring members independently selected from O, N, and S.
  • any ring-forming N in a heteroaryl moiety can be an N-oxide.
  • the heteroaryl has 4-6 ring atoms and 1 , 2, 3 or 4 heteroatom ring members independently selected from O, N, and S.
  • the heteroaryl has 5-6 ring atoms and 1 or 2 heteroatom ring members independently selected from O, N, and S.
  • the heteroaryl is a five-membered or six-membered heteroaryl ring.
  • exemplary heteroaryl groups include, but are not limited to, thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, 1,2,3-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3- oxadiazolyl, 1,2,4-triazolyl, 1,2,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-triazolyl, 1,3,4-thiadiazolyl, 1,3,4-oxadiazolyl, pyridyl, pyrazinyl, pyrimidinyl, triazinyl, and pyridazinyl.
  • heterocycloalkyl refers to non-aromatic heterocycles having one or more ring-forming heteroatoms selected from O, N, and S.
  • Example heterocycles include, but are not limited to, pyrrolidin-2-one, l,3-isoxazolidin-2-one, pyranyl, tetrahydropuran, oxetanyl, azetidinyl, morpholino, thiomorpholino, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl, pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, imidazolidinyl, azepanyl, benzazapene, and the like.
  • the heterocycloalkyl group can be attached through a ring-forming carbon atom or a ring-forming heteroatom.
  • the heterocyclo alkyl is a 3-6 membered heterocycloalkyl having 1, 2, 3 or 4 heteroatom ring members independently selected from O, N, and S.
  • the heterocycloalkyl is a 5-6 membered heterocycloalkyl.
  • the term "compound" as used herein is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures depicted. Compounds herein identified by name or structure as one particular tautomeric form are intended to include other tautomeric forms unless otherwise specified.
  • Tautomeric forms result from the swapping of a single bond with an adjacent double bond together with the concomitant migration of a proton.
  • Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge.
  • Example prototropic tautomers include ketone - enol pairs, amide - imidic acid pairs, lactam - lactim pairs, enamine - imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, for example, 1H- and 3H-imidazole, 1H-, 2H- and 4H- 1,2,4-triazole, 1H- and 2H- isoindole, and 1H- and 2H-pyrazole.
  • Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.
  • All compounds, and pharmaceutically acceptable salts thereof, can be found together with other substances such as water and solvents (e.g. hydrates and solvates) or can be isolated.
  • preparation of compounds can involve the addition of acids or bases to affect, for example, catalysis of a desired reaction or formation of salt forms such as acid addition salts.
  • Example acids can be inorganic or organic acids and include, but are not limited to, strong and weak acids.
  • Some example acids include hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, ?-toluenesulfonic acid, 4- nitrobenzoic acid, methanesulfonic acid, benzenesulfonic acid, trifluoroacetic acid, and nitric acid.
  • Some weak acids include, but are not limited to acetic acid, propionic acid, butanoic acid, benzoic acid, tartaric acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, and decanoic acid.
  • Example bases include lithium hydroxide, sodium hydroxide, potassium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate, and sodium bicarbonate.
  • Some example strong bases include, but are not limited to, hydroxide, alkoxides, metal amides, metal hydrides, metal dialkylamides and arylamines, wherein; alkoxides include lithium, sodium and potassium salts of methyl, ethyl and t-butyl oxides; metal amides include sodium amide, potassium amide and lithium amide; metal hydrides include sodium hydride, potassium hydride and lithium hydride; and metal dialkylamides include lithium, sodium, and potassium salts of methyl, ethyl, n-propyl, wo-propyl, n-butyl, tert-butyl, trimethylsilyl and cyclohexyl substituted amides.
  • the compounds provided herein, or salts thereof are substantially isolated.
  • substantially isolated is meant that the compound is at least partially or substantially separated from the environment in which it was formed or detected.
  • Partial separation can include, for example, a composition enriched in the compounds provided herein.
  • Substantial separation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of the compounds provided herein, or salt thereof. Methods for isolating compounds and their salts are routine in the art.
  • the present application further provides methods of treating a disease associated with KSR (e.g., upregulation of KSR expression) in a patient in need thereof.
  • the disease associated with KSR is a disease mediated by KSR.
  • the term "patient” refers to any animal, including mammals, for example, mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, primates, and humans.
  • the patient is a human.
  • the disease is selected from the group consisting of cancer, diabetes, obesity, a neurological disease, a skin disorder, an adverse effect associated with aging, a cardiovascular disease, organ transplant rejection, and graft versus host disease.
  • the method comprises administering to a patient in need thereof a therapeutically effective amount of a compound provided herein (e.g., a compound of Formula I, a compound of Formula II, a compound of Formula III, or a compound selected from group the consisting of compounds (1)-(114)) or a pharmaceutically acceptable salt thereof.
  • a compound provided herein e.g., a compound of Formula I, a compound of Formula II, a compound of Formula III, or a compound selected from group the consisting of compounds (1)-(114)
  • the compound is a compound of Formula I, or a pharmaceutically acceptable salt thereof.
  • the compound is a compound of Formula II, or a pharmaceutically acceptable salt thereof.
  • the compound is a compound of Formula III, or a pharmaceutically acceptable salt thereof.
  • the compound is selected from the group consisting of compounds (1)-(114), or a pharmaceutically acceptable salt thereof.
  • the compound is 6,7- dimethoxy-N-(2-methyl-4-phenoxyphenyl)quinazolin-4-
  • the compound is 6,7- dimethoxy-N-(2-methyl-4-phenoxyphenyl)quinazolin-4-amine 2,2,2-trifluoroacetate (21 -TFA). In some embodiments, the compound is 6,7-dimethoxy-N-(2-methyl-4- phenoxyphenyl)quinazolin-4-amine hydrochloride (21-HCI).
  • the disease is cancer.
  • the cancer is selected from the group consisting of breast cancer, prostate cancer, esophageal cancer, colon cancer, endometrial cancer, brain cancer, bladder cancer, skin cancer, cancer of the uterus, cancer of the ovary, lung cancer, pancreatic cancer, renal cancer, prostate cancer, gastric cancer, stomach cancer, and hematological cancer.
  • the lung cancer is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, and lung carcinoid tumor.
  • the hematological cancer is selected from the group consisting of leukemia, lymphoma, and multiple myeloma.
  • the hematological cancer is acute myeloblastic leukemia, chronic myeloid leukemia, B cell lymphoma, chronic lymphocytic leukemia (CLL), Non-Hodgkins lymphoma, hairy cell leukemia, Mantle cell lymphoma, Burkitt lymphoma, small lymphocytic lymphoma, follicular lymphoma, lymphoplasmacytic lymphoma, extranodal marginal zone lymphoma, activated B-cell like (ABC) diffuse large B cell lymphoma, or germinal center B cell (GCB) diffuse large B cell lymphoma.
  • BCBC activated B-cell like
  • GCB germinal center B cell
  • the leukemia is selected from the group consisting of acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (AML), chronic myeloid leukemia (CML), hairy cell leukemia, T-cell prolymphocytic leukemia, juvenile myelomonocytic leukemia, and follicular lymphoma.
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia
  • AML acute myelogenous leukemia
  • CML chronic myeloid leukemia
  • hairy cell leukemia T-cell prolymphocytic leukemia
  • juvenile myelomonocytic leukemia juvenile myelomonocytic leukemia
  • follicular lymphoma follicular lymphoma
  • the lymphoma is Hodgkin's lymphoma or non- Hodgkin's lymphoma (NHL).
  • the non-Hodgkin lymphoma is selected from relapsed NHL, refractory NHL, and recurrent follicular NHL.
  • the disease is diabetes or obesity. In some embodiments, the disease is diabetes or obesity.
  • the disease is a neurological disease (e.g., a mood disorder, age onset proteotoxic disease (i.e., Alzheimer's disease).
  • the disease is a skin disorder (e.g., acne vulgaris).
  • the disease is an adverse effect associated with aging (e.g., cellular degradation associated with aging).
  • the disease is a cardiovascular disease (e.g., cardiac hypertrophy or cardiac disease).
  • the disease is organ transplant rejection.
  • the disease is graft versus host disease.
  • the method further comprises administering an additional therapeutic agent to the patient.
  • additional therapeutic agents include, but are not limited to antibiotic agents, antiviral agents, antifungal agents, anesthetics (e.g., for use in combination with a surgical procedure), anti-inflammatory agents, anti-allergic agents, and chemotherapeutic agents.
  • the additional therapeutic agent comprises radiation therapy.
  • a compound provided herein is administered in combination with an additional therapeutic agent during a surgical procedure.
  • the additional therapeutic agent is administered simultaneously with a compound provided herein. In some embodiments, the additional therapeutic agent is administered after administration of a compound provided herein. In some embodiments, the additional therapeutic agent is administered prior to administration of a compound provided herein.
  • the additional therapeutic agent is selected from the group consisting of an antibiotic agent, an antiviral agent, an antifungal agent, an anesthetic, or an anti-inflammatory agent (e.g., steroidal and non-steroidal antiinflammatories), and an anti-allergic agent.
  • an antibiotic agent e.g., an antibiotic agent, an antiviral agent, an antifungal agent, an anesthetic, or an anti-inflammatory agent (e.g., steroidal and non-steroidal antiinflammatories), and an anti-allergic agent.
  • Suitable medicaments include, but are not limited to, coriticosteroids such as dexamethasone or prednisone, aminoglycosides such as amikacin, gentamycin, tobramycin, streptomycin, netilmycin, and kanamycin; fluoroquinolones such as ciprofloxacin, norfloxacin, ofloxacin, trovafloxacin, lomefloxacin, levofloxacin, and enoxacin; naphthyridine; sulfonamides; polymyxin; chloramphenicol; neomycin; paramomycin; colistimethate; bacitracin; vancomycin; tetracyclines; rifampin and its derivatives ("rifampins”); cycloserine; beta-lactams; cephalosporins; amphotericins; fluconazole; flucytosine; natamycin; miconazole; keto
  • ketorolac ketorolac
  • suprofen cromolyn
  • lodoxamide levocabastin
  • naphazoline antazoline
  • pheniramine or azalide antibiotic.
  • the additional therapeutic agent is a chemotherapeutic agent.
  • the additional therapeutic agent is a kinase inhibitor (e.g., KSR, MEK, Bcr-Abl, Flt-3, EGFR, HER2, JAK, c-MET, VEGFR, PDGFR, PI3K, cKit, IGF-1R, RAF, FAK, Akt mTOR, PIM, and AKT).
  • the additional therapeutic agent can be a MEK inhibitor or a KSR inhibitor.
  • the additional therapeutic agent is selected from the group consisting of a cytostatic agent, cisplatin, doxorubicin, taxol, etoposide, irinotecan, topotecan, paclitaxel, docetaxel, epothilones, tamoxifen, 5-fluorouracil, methotrexate, temozolomide, cyclophosphamide, tipifarnib, gefitinib, erlotinib hydrochloride, antibodies to EGFR, imatinib mesylate, gemcitabine, uracil mustard, chlormethine, ifosfamide, melphalan, chlorambucil, pipobroman, triethylenemelamine,
  • a cytostatic agent cisplatin
  • doxorubicin taxol
  • etoposide etoposide
  • irinotecan topotecan
  • paclitaxel docetaxel
  • the additional therapeutic agent is a MEK inhibitor.
  • the MEK inhibitor is a MEK1 and MEK2 inhibitor.
  • the MEK inhibitor is a MEK1 inhibitor.
  • the MEK inhibitor is a MEK2 inhibitor.
  • the MEK inhibitor is selected from the group consisting of trametinib, selumetinib, binimetinib, refametinib, pimasertib, cobimetinib, AZD8330, R04987655, R05126766, WX-554, E6201, MSC1936369B, PD-325901, CI-1040, RDEA1 19, CH5126766, GDC-0623, G-573, TAK-733, TAK-133, CI-1 040/PDl 84352, AZD6244, PD318088, PD98059, PD334581, RDEAl 19, 6-methoxy-7-(3-morpholin-4-yl-propoxy)-4-(4-phenoxy- phenylamino)quinoline-3-carbonitrile, and 4-[3-Chloro-4-(l-methyl-lH-imidazol-2- ylsulfanyl)phenylamin
  • the present application further provides a method of treating a disease provided herein (e.g., cancer, diabetes, obesity, an adverse effect associated with aging, a neurological disease, a skin disorder, a cardiovascular disease, organ transplant rejection, and graft versus host disease) in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound provided herein (e.g. , a compound of Formula I, a compound of Formula II, a compound of Formula III, or a compound selected from group the consisting of compounds (1)-(114)) or a pharmaceutically acceptable salt thereof, and an additional therapeutic agent provided herein.
  • a compound provided herein e.g. , a compound of Formula I, a compound of Formula II, a compound of Formula III, or a compound selected from group the consisting of compounds (1)-(114)
  • the compound is 6,7- dimethoxy-N-(2-methyl-4-phenoxyphenyl)quinazolin-4-amine (21), or a
  • the compound is 6,7- dimethoxy-N-(2-methyl-4-phenoxyphenyl)quinazolin-4-amine 2,2,2-trifluoroacetate (21-TFA). In some embodiments, the compound is 6,7-dimethoxy-N-(2-methyl-4- phenoxyphenyl)quinazolin-4-amine hydrochloride (21-HC1). In some embodiments, the disease is cancer. In some embodiments, the disease is diabetes or obesity. In some embodiments, the disease is an adverse effect associated with aging (e.g., cellular degradation associated with aging). In some embodiments, the disease is a neurological disease (e.g., a mood disorder, age onset proteotoxic disease (i.e.,
  • the disease is a skin disorder (e.g., acne vulgaris).
  • the disease is a cardiovascular disease (e.g., cardiac hypertrophy or cardiac disease).
  • the disease is organ transplant rejection.
  • the disease is graft versus host disease.
  • the additional therapeutic agent is a MEK inhibitor.
  • the MEK inhibitor is a MEKl and MEK2 inhibitor.
  • the MEK inhibitor is a MEKl inhibitor.
  • the MEK inhibitor is a MEK2 inhibitor.
  • the MEK inhibitor is selected from the group consisting of trametinib, selumetinib, binimetinib, refametinib, pimasertib, cobimetinib, AZD8330, R04987655, R05126766, WX-554, E6201, MSC1936369B, PD-325901, CI-1040, RDEA119, CH5126766, GDC-0623, G-573, TAK-733, TAK-133, CI-1 040/PD1 84352, AZD6244, PD318088, PD98059, PD334581, RDEA1 19, 6-methoxy-7-(3-morpholin-4-yl-propoxy)-4-(4-phenoxy- phenylamino)quinoline-3-carbonitrile, and 4- [3 -Chloro-4-( 1 -methyl- 1 H-imidazol-2- ylsulfanyl)pheny
  • the present application further provides a method of treating a disease provided herein (e.g., cancer, diabetes, obesity, adverse effect associated with aging, a neurological disease, a skin disorder, a cardiovascular disease, organ transplant rejection, and graft versus host disease) in a patient in need thereof, comprising:
  • a compound provided herein e.g., a compound of Formula I, a compound of Formula II, a compound of Formula III, or a compound selected from group the consisting of compounds (1)-(114)
  • the compound is 6,7- dimethoxy-N-(2-methyl-4-phenoxyphenyl)quinazolin-4-amine (21), or a
  • the compound is 6,7- dimethoxy-N-(2-methyl-4-phenoxyphenyl)quinazolin-4-amine 2,2,2-trifluoroacetate (21'TFA). In some embodiments, the compound is 6,7-dimethoxy-N-(2-methyl-4- phenoxyphenyl)quinazolin-4-amine hydrochloride (21-HCl).
  • the disease associated with KSR is a disease mediated by KSR. In some embodiments, the disease is associated with KSRl and KSR2. In some embodiments, the disease is associated with KSRl or KSR2. In some embodiments, the disease is associated with KSRl. In some embodiments, the disease is associated with KSR2. In some embodiments, the disease is mediated by KSRl and KSR2. In some embodiments, the disease is mediated by KSRl or KSR2. In some embodiments, the disease is mediated by KSRl . In some embodiments, the disease is mediated by KSR2.
  • the disease is cancer. In some embodiments, the disease is diabetes or obesity. In some embodiments, the disease is an adverse effect associated with aging (e.g., cellular degradation associated with aging). In some embodiments, the disease is a neurological disease (e.g., a mood disorder, age onset proteotoxic disease (i.e., Alzheimer's disease). In some embodiments, the disease is a skin disorder (e.g., acne vulgaris). In some embodiments, the disease is a
  • cardiovascular disease e.g., cardiac hypertrophy or cardiac disease.
  • the disease is organ transplant rejection.
  • the disease is graft versus host disease.
  • the KSR comprises KSRl . In some embodiments, the KSR comprises KSR2. In some embodiments, the KSR comprises KSRl and KSR2. In some embodiments, the KSR is KSRl . In some embodiments, the KSR is KSR2.
  • the method further comprises administering an additional therapeutic agent provided herein.
  • the additional therapeutic agent is a chemotherapeutic agent.
  • the additional therapeutic agent is a MEK inhibitor.
  • the MEK inhibitor is a MEK1 and MEK2 inhibitor.
  • the MEK inhibitor is a MEK1 inhibitor.
  • the MEK inhibitor is a MEK2 inhibitor.
  • the MEK inhibitor is selected from the group consisting of trametinib, selumetinib, binimetinib, refametinib, pimasertib, cobimetmatib, AZD8330, R04987655, R05126766, WX-554, E6201, MSC1936369B, PD-325901, CI-1040, RDEA1 19, CH5126766, GDC-0623, G-573, TAK-733, TAK-133, CI-1 040/PDl 84352, AZD6244, PD318088, PD98059, PD334581, RDEA1 19, 6-methoxy-7-(3-morpholin-4-yl-propoxy)-4-(4-phenoxy- phenylamino)quinoline-3-carbonitrile, and 4-[3-Chloro-4-(l-methyl-lH-imidazol-2- ylsulfanyl)phenylamino
  • the present application further provides a method of inhibiting KSR in a cell, comprising contacting the KSR with a compound provided herein (e.g. , a compound of Formula I, a compound of Formula II, a compound of Formula III, or a compound selected from group the consisting of compounds (1)-(114)) or a pharmaceutically acceptable salt thereof.
  • a compound provided herein e.g. , a compound of Formula I, a compound of Formula II, a compound of Formula III, or a compound selected from group the consisting of compounds (1)-(114)
  • the compound is 6,7-dimethoxy-N-(2- methyl-4-phenoxyphenyl)quinazolin-4-amine (21), or a pharmaceutically acceptable salt thereof.
  • the compound is 6,7-dimethoxy-N-(2-methyl-4- phenoxyphenyl)quinazolin-4-amine 2,2,2-trifluoroacetate (21-TFA).
  • the compound is 6,7-dimethoxy-N-(2-
  • the KSR comprises KSR1. In some embodiments, the KSR comprises KSR2. In some embodiments, the KSR comprises KSR1 and KSR2. In some embodiments, the KSR is KSR1. In some embodiments, the KSR is KSR2.
  • the present application further provides a method of inhibiting a KSR and a MEK in a cell, comprising contacting the cell with a compound provided herein (e.g., a compound of Formula I, a compound of Formula II, a compound of Formula III, or a compound selected from group the consisting of compounds (1)-(114)) or a pharmaceutically acceptable salt thereof.
  • a compound provided herein e.g., a compound of Formula I, a compound of Formula II, a compound of Formula III, or a compound selected from group the consisting of compounds (1)-(114)
  • the compound is 6,7- dimethoxy-N-(2-methyl-4-phenoxyphenyl)quinazolin-4-amine (21), or a
  • the compound is 6,7- dimethoxy-N-(2-methyl-4-phenoxyphenyl)quinazolin-4-amine 2,2,2-trifluoroacetate (21-TFA). In some embodiments, the compound is 6,7-dimethoxy-N-(2-methyl-4- phenoxyphenyl)quinazolin-4-amine hydrochloride (21-HCl).
  • the KSR comprises KSRl . In some embodiments, the KSR comprises KSR2. In some embodiments, the KSR comprises KSRl and KSR2. In some embodiments, the KSR is KSRl . In some embodiments, the KSR is KSR2.
  • the MEK comprises MEKl. In some embodiments, the MEK comprises MEK2. In some embodiments, the MEK comprises MEKl and MEK2. In some embodiments, the MEK is MEKl . In some embodiments, the MEK is MEK2.
  • the method further comprises contacting the cell with a MEK inhibitor.
  • the MEK inhibitor is a MEKl and MEK2 inhibitor.
  • the MEK inhibitor is a MEKl inhibitor.
  • the MEK inhibitor is a MEK2 inhibitor.
  • the MEK inhibitor is selected from the group consisting of trametinib, selumetinib, binimetinib, refametinib, pimasertib, cobimetinib, AZD8330, R04987655,
  • phrases "pharmaceutically acceptable amount” or “therapeutically effective amount” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • a “pharmaceutically acceptable amount” or “therapeutically effective amount” refers to the amount of active compound or pharmaceutical agent that elicits the biological or medicinal response that is being sought in a tissue, system, animal, individual or human by a researcher, veterinarian, medical doctor or other clinician.
  • treating refers to one or more of (1) inhibiting the disease; for example, inhibiting a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., arresting further development of the pathology and/or symptomatology); and (2) ameliorating the disease; for example, ameliorating a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., reversing the pathology and/or symptomatology) such as decreasing the severity of disease or reducing or alleviating one or more symptoms of the disease.
  • the pH of aqueous solutions was estimated using pH paper. Vacuum filtrations were carried out using a house vacuum line ( ⁇ 100 torr). In the individual procedures, the phrases “concentrated under vacuum” and “concentrated to dryness” mean that solvent was removed on a rotary evaporator using a diaphragm pump (with an automatic vacuum regulator) and then remaining traces of volatiles were removed on a high-vacuum ( ⁇ 1 torr) oil pump. Unless specified otherwise, the term “flask” refers to the round-bottomed variety.
  • A375, HCT-116, and A549 cells were obtained from the American Type Culture Collection.
  • SK-MEL-239 and 293H cells were rationally provided by the Poulikakos Lab at the Icahn School of Medicine at Mount Sinai.
  • Cell lines were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum supplement with penicillin/streptomycin.
  • Antibodies for detection of KSR1 (Product number: 4640), Flag-tag (Product number: 8146), GFP (Product number: 2555), pMEK (Product number: 9121), pERK (Product number: 9101), and MEK (Product number: 9122) were obtained from Cell Signaling. Western blotting was performed as described herein. For primary antibody detection, blots were washed several times and probed for 1 hr with anti-Mouse-HRP or anti-Rabbit-HRP (Cell Signaling) in 5% milk TBST.
  • KSR1-FLAG (Addgene ID:25970) and MEKl-GFP (Addgene ID: 14746) plasmids were obtained from Addgene.
  • the plasmids for expression of RAF was generated by DNA 2.0. Constructs harboring mutations were generated by site- directed mutagenesis (Agilent). DNA and siRNA transfections were carried out using Fugene 6 (Promega) and Dharmafect (GE Dharmacon) reagents in accordance with the manufacturer's recommendations.
  • Trametinib and dabrafenib were obtained from Selleck Chemicals. All compounds were dissolved in DMSO and subsequently diluted for biochemical and cellular experiments.
  • Example 1 The hydrochloride salt of Example 1 (i.e., compound 2-HCl) was suspended in a mixture of water and 95:5 CH 2 Cl 2 /MeOH, and then the pH of the aqueous phase was adjusted to 7-8 with a saturated solution of NaHC0 3 . The mixture was transferred to a separatory funnel, the layers were separated and the aqueous phase was extracted with a 95:5 mixture of CH2Cl 2 /MeOH (X2). The organic extracts were pooled, dried (Na 2 S04), filtered and concentrated to dryness: 3 ⁇ 4 NMR (400 MHz, DMSO-de) ⁇
  • Table 1 shows Compounds 1-55, which were prepared according to one or more of the procedures provided herein, substituting the appropriate starting materials, and the corresponding characterization data. Table 1.
  • the ATP-biotin assay was performed in buffer containing 25 mM Tris pH 7.5,
  • hKSR2-rMEKl 150 mM NaCl, 10 mM MgCl 2 , and 2% DMSO.
  • Purified hKSR2-rMEKl was assayed at 0.5 ⁇ .
  • hKSR2-rMEKl was pre-incubated with 20 ⁇ of the indicated compounds for 15 minutes.
  • ATP-biotin (Pierce Cat. #88310) was added to a final concentration of 2 ⁇ . Reactions were incubated at room temperature for 5 minutes before being stopped by the addition of 6X SDS loading dye. Samples were then electrophoresed on a 4-15% Tris-HCl SDS gradient gel, transferred to nitrocellulose membranes and blotted with Strepdavidin-HRP.
  • Enhanced hKSR2-rMEKl 150 mM NaCl, 10 mM MgCl 2 , and 2% DMSO.
  • chemiluminescence signals corresponding to labeling on KSR2 and MEKl were visualized and quantified using the Biodoc system (Biorad). Relative signals of Streptavidin HRP on bands corresponding to KSR2 and MEKl in the presence of compounds relative to DMSO controls were used to determine 'Percent Inhibition of ATP Probe Labeling'. Control experiments using ATP as a competitor were used to determine that the ATP-biotin probe specifically labels the active-site of KSR2 and MEKl. Representative results of the ATP-biotin assay are shown in FIGs. 1-2.
  • ATP blotin transfers a desthio-biotin group via a reactive acyl-phosphate linkage onto active-site lysines when bound to either KSR2 or MEKl (Patricelli et al, Biochemistry, 2007, 46:350-358). It was confirmed that this label leads to a covalent attachment of desthiobiotin on KSR through detection with Streptavidin-HRP and intact mass spectrometry, the latter of which confirmed the addition of a two or one desthiobiotin groups (equivalent to a mass increase of 196.1 Da per desthiobiotin group) onto both KSR2 and MEKl, respectively, under non-saturating conditions.
  • ATP bl0tm labeling competition screen samples were applied to a 4— 20% Tris-HCl gel, separated, and then transferred to a nitrocellulose membrane. The membrane was blocked with 5% bovine serum albumin diluted in TBS-T for 30 minutes and subsequently probed with the PierceTM High Sensitivity Streptavidin- F£RP. After several washes, the membranes were visualized using enhanced chemiluminescence on a Biodoc (Biorad). ICso values were determined under similar assay conditions with slight modifications: 0.1 ⁇ KSR2:MEK1 was pre-incubated with a dose-range of compounds (27 nM to 20 ⁇ in three-fold dilutions prior to the addition of ATP bl0tm . Assays were quenched and analysed similarly to as described above. Sigmoidal dose response curves were used to derive ICso values in Prism 6.0 (Graphpad). Assays were completed in triplicate.
  • KSR2(KD) human KSR2 kinase domain
  • MEKl(35-393) the final size exclusion chromatography step of the human KSR2 kinase domain (KSR2(KD)):MEKl(35-393) purification was performed in buffer containing 15 mM Bis-Tris pH 6.5, 150 NaCl, 1 mM TCEP, and 5 mM DTT.
  • the KSR2(KD):MEKl(35-393) complex was concentrated to 10 mg/mL and incubated with 500 ⁇ Compound 21 » TFA. Aggregates were removed through centrifugation at 14,000 g for 10 minutes. Over a thousand conditions were screen using in-house and sparse matrix screens via sitting-drop methods at 4, 10, and 22 °C.
  • KSR2(KD):MEK1 :ATP crystal structure (PDB code: 2Y4I). Model building and refinement was performed with Coot and Phenix (see e.g. , Emsley et al, Acta crystallographica. Section D, Biological crystallography, 2010, 66:486-501; and Adams et al, Acta crystallographica. Section D, Biological crystallography, 2010, 66:213-221). Crystals for the ATP and Compound 21 bound KSR2:MEK1 complexes share similar unit cell dimensions, space group symmetry, and X-ray diffraction properties.
  • KSR2 binds MEK1 in a 1 :1 fashion within a quaternary arrangement that is nearly identical to the ATP-bound state of KSR2:MEK1 complexes (see e.g., Brennan et al, Nature, 2011, 472:366-369).
  • KSR2 and MEK1 bind via a face-to- face arrangement mediated in large part through reciprocal helix aG and activation segment interactions, and KSR2 homo-dimerizes through the N-lobe along a crystallographic 2-fold symmetry axis producing a hetero-tetramer of KSR2:MEK1 dimers.
  • Differences between the ATP- and Compound 21 bound states include variations in ligand occupancy and conformational changes at the level of secondary structural elements that are localized to KSR2.
  • the MEK1 activation segment containing two phospho-regulatory sites (Ser218 and Ser222) necessary for MEK1 activation, is buried within the KSR2:MEK1 interaction interface.
  • the KSR2:MEK1 Compound 21 structure revealed electron density for a portion of KSR2 that was not previously modeled, and therefore disordered in the ATP-bound complex, as shown in FIG. 6.
  • This region encompassing residues Leu809 to Gln814, forms an extension of the activation segment C-terminal to the conserved DFG motif, and forms an anti-parallel beta-strand with residues Asp820 to Gln825 in KSR2.
  • Asp820 through to Gln825 directly engages the activation segment of MEK1 burying the Ser218-Ser222 region, presumably shielding this segment of MEK from promiscuous phosphorylation.
  • B-RAF was purified as previously described with slight modification (see e.g., Tsai et al, Proceedings of the National Academy of Sciences of the United States of America, 2008, 105:3041-3046).
  • a variant of B-RAF was identified that could be expressed in high yield from E.Coli and which also retained near wild-type levels of kinase activity towards MEK1.
  • indicated concentrations of B-RAF and 1 ⁇ KSR2-MEK1 or 1 ⁇ MEK1 proteins were pre- incubated with 10 ⁇ of indicated compounds for 15 minutes in buffer containing 25 mM Tris pH 7.5, 150 mMNaCl, 10 mM MgC12, and 2% DMSO.
  • RAF can function as both an allosteric regulator of KSR and catalytic enzyme responsible for MEK phosphorylation (see e.g. , Farrar et al, Nature, 1996, 383: 178- 181; Brennan et al, Nature, 2011, 472:366-369; Huang et al, Proceedings of the National Academy of Sciences of the United States of America, 1993, 90:10947-
  • bio-layer inferometry was utilized to monitor real-time association and dissociation of KSR2:MEK1 or free MEK1 to a sensor tethered with immobilized B-RAF.
  • KSR2:B-RAF dimerization a mutation in B-RAF F667E was identified that eliminates binding to free MEK but not KSR2-MEK1 complexes, as shown in FIGs. 7C-7D.
  • KSR2:MEK1 interacted in a 1 :1 fashion with the B-RAF F667E mutant with a KD of 1.99 +/- 0.09 ⁇ .
  • this binding constant represents the dimerization constant for KSR2:B-RAF as it nearly matches previously published B-RAF:B-RAF dimerization values (see e.g., Rajakulendran et al, Nature, 2009, 461 :542-545).
  • Ser218/Ser222 by RAF could be enhanced at least 10-fold in the presence of KSR, as shown in FIG. 8.
  • KSR-stimulated MEK phosphorylation by RAF was significantly reduced by addition of Compound 21, as shown in FIGs. 9A-9C.
  • FIGs. 9A-9C KSR-stimulated MEK phosphorylation by RAF
  • Compound 21 was essentially inactive when KSR was absent or when the KSR2 A690F mutant was used for in vitro assays. Furthermore, Compound 21 lacked detectable inhibitory activity against purified forms of RAF, MEK, or ERK. Finally, Compound 21 was found to mimic the effects of RNAi based knockdown of KSRI in growth- factor stimulated cells, as shown in FIGs. 10A-10B.
  • Model cell lines A375 (BRAF V600E positive) and HCT-116 (KRas G12D positive) were treated with the Compound 21 in combination with a MEK inhibitor, trametinib.
  • a reduction in MAPK pathway readouts based on phospho-antibodies was observed when trametinib was combined with the Compound 21, as shown in FIG. 11.
  • Model cell lines HCT-116, A549, and LOVO were plated at a density of 500 cells per well in a 96 well plate. Cell were then treated with increasing concentrations (0.1 ⁇ , 0.3 ⁇ , 1 ⁇ , 3 ⁇ , 10 ⁇ ) of Compound 21 ⁇ i.e., APS-2-79) for 72 hours. Cell viability was analyzed using the flourescent resazurin assay. Resazurin was added to each well to a final concentration of 0.02 mg/mL. After 3 h of incubation at 37 °C, cell viability was determined by measuring flouresence with an excitation 560 nM/590 nM emission filter set. FIGs. 15A-15C show the results of the in vitro cell assays using Compound 21.
  • BLISS index was calculated by determining the difference between the expected combination value (the product of the viability ratio of drugl alone and drug2 alone) and the observed combination value (the viability ratio observed from combination treatment with drugl and drug2).
  • the viability ratio represented the viability observed for treated (inhibitors) relative to controls (DMSO). The sum of these values across each 8 x 5 concentration matrix is presented in FIG. 12.
  • the RAF- immobilized sensor heads were incubated with 5 different concentrations of purified KSR2:MEK1 complex (range: 625 nM to 10 ⁇ in two-fold dilutions). Association and dissociation was monitored over 660 and 840 seconds, respectively. Data sets were normalized and analysed using global fit binding models (Fortebio software). Kon and Koff were used to derive the KD values in FIGs. 7A-7G. OTHER EMBODIMENTS

Abstract

La présente invention concerne des antagonistes de la kinase suppresseur de Ras (KSR). L'invention concerne également des compositions pharmaceutiques comprenant lesdits inhibiteurs de la kinase KSR ainsi que des méthodes de traitement du cancer.
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